Transcription and replication are fundamental physiological processes, yet paradoxically they both pose a threat to the stability of the genome. Highly transcribed DNA loci are associated with increased levels of recombination and mutagenesis, and replication forks encounter DNA lesions and other types of replication stress every cell cycle. Physical conflicts between the protein complexes involved in replication and transcription also threaten genome stability. Recent studies suggest that the deleterious effects of transcription could be mediated during S phase by R-loops, three-stranded nucleic acid structures containing an RNA-DNA hybrid and region of single-stranded DNA. R-loops occur throughout the genome of mammalian cells and regulate various aspects of gene expression, but in some circumstances their formation is correlated with DNA damage. How R-loops affect replication and transcription, how they are resolved, and how they ultimately lead to DNA damage are unresolved questions. The long-term goal of this research program is to understand how cells distinguish and process regulatory and deleterious R-loops, and how this is perturbed in human disease. It is hypothesized that R-loops are dynamic structures, but that they accumulate to toxic levels when transcription, RNA processing or DNA replication is perturbed. Under these conditions, accumulated R-loops pose threats to replication and transcription and cause DNA damage, ultimately resulting in genome instability. The objective of this application is to define how R-loops are recognized and processed by the cell, and how defects in these pathways affect DNA replication, transcription and genome stability. In the first aim, the processing of R-loops by cellular nucleases involved in DNA repair will be explored. In the second aim, a novel system recently developed will be utilized to study collisions between the replication and transcription machineries in cells, and th impact of an R-loop on these collisions. Finally, in the third aim, the effects of R- loop processing on transcription, replication and genome stability will be probed. These studies will take advantage of cutting-edge genomic approaches to map the spatial distribution of R-loops, transcriptional activity and double-strand breaks in the genome.